Traditional Chinese medicine is one of the oldest medical systems in the world and has its unique principles and theories in the prevention and treatment of human diseases, which are achieved through the interactions of different types of materia medica in the form of Chinese medicinal formulations. GZZSZTW, a classical and effective Chinese medicinal formulation, was designed and created by professor Bailing Liu who is the only national medical master professor in the clinical research field of traditional Chinese medicine and skeletal diseases. GZZSZTW has been widely used in clinical settings for several decades for the treatment of joint diseases. However, the underlying molecular mechanisms are still largely unknown. In the present study, we performed quantitative proteomic analysis to investigate the effects of GZZSZTW on mouse primary chondrocytes using state-of-the-art iTRAQ technology. We demonstrated that the Chinese medicinal formulation GZZSZTW modulates chondrocyte structure, dynamics, and metabolism by controlling multiple functional proteins that are involved in the cellular processes of DNA replication and transcription, protein synthesis and degradation, cytoskeleton dynamics, and signal transduction. Thus, this study has expanded the current knowledge of the molecular mechanism of GZZSZTW treatment on chondrocytes. It has also shed new light on possible strategies to further prevent and treat cartilage-related diseases using traditional Chinese medicinal formulations.